Development of a high resolution melting method for the detection of genetic variations in Long QT Syndrome

Millat, Gilles; Chanavat, Valérie; Crehalet, Hervé; Rousson, Robert

doi:10.1016/j.cca.2010.09.013

Cited by 11 publications

(10 citation statements)

References 15 publications

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“…In addition to the LTCC-related genes (CACNA1C, CACNB2b), we screened SCN5A, KCNQ1, KCNH2, KCNE1-3, KCNE5, 17 SCN3B 18 and KCNJ8 19-21 using a high-resolution melting method (HRM) 22 or denaturing high-performance liquid chromatography (dHPLC; WAVE system Model 3500, Transgenomic, Omaha, NE, USA) and subsequent direct sequencing. Briefly, the coding exons of genes were amplified using primers as previously reported.…”

Section: Gene Scanningmentioning

confidence: 99%

L-Type Calcium Channel Mutations in Japanese Patients With Inherited Arrhythmias

Fukuyama

Ohno

Wang

et al. 2013

Circ J

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Section: Gene Scanningmentioning

confidence: 99%

L-Type Calcium Channel Mutations in Japanese Patients With Inherited Arrhythmias

Fukuyama

Ohno

Wang

et al. 2013

Circ J

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“…It has been applied to detect mutations of KCNQ1, KCNH2 and SCN5A. 10,11,18) Millat, et al reported 2 exons (exon 1 and 16) of the KCNQ1 gene did not obtain a sufficient amplicon quality for subsequent HRM analysis and Farrugia, et al found 3 exons (exons 1, 13 and 16). In the present study, we showed that exons 1 and 16 were not suitable for HRM analysis and our results were similar to those of Millat, et al We used the same instrument with the same software and same PCR kit as Farrugia, et al One of the hypotheses that might explain the difference in the results for exon 13 between our study and that of Farrugia, et TM PCR System.…”

Section: Discussionmentioning

confidence: 99%

“…Examples of such methods are direct sequencing, denaturing high performance liquid chromatography (DHPLC), single strand conformational polymorphism analysis (SSCP), HRM analysis, and next-generation sequencing (NGS). 3,[8][9][10][11][12] Some methods for large-scale detection of the 3 major LQTScausing gene mutations are expensive and technically timeconsuming. Since 2002, HRM analysis has represented the next generation of mutation scanning technology and offers considerable time and cost savings over those methods.…”

Section: Ong Qt Syndrome (Lqts) Is a Congenital Arrhyth-mentioning

confidence: 99%

Mutation Analysis of KCNQ1, KCNH2 and SCN5A Genes in Taiwanese Long QT Syndrome Patients

et al. 2015

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SummaryLong QT syndrome (LQTS) is a genetic cardiac disease. Gene mutation affects the structure or function of ion channels that are associated with a high risk of sudden death. The goal of this study was to determine the frequency of KCNQ1, KCNH2, and SCN5A mutations in LQTS in a Taiwanese population. Genomic DNA was extracted from peripheral blood samples obtained from 5 patients with LQTS and the family members of 3 LQTS patients. High resolution melting (HRM) analysis and direct DNA sequencing were used to characterize the KCNQ1, KCNH2, and SCN5A genetic variations. HRM analysis was successfully optimized for 14 of the 16 exons of the KCNQ1, 5 of the 15 exons of the KCNH2, and 23 of the 27 exons of the SCN5A. HRM and direct DNA sequencing was applied to the cohort of 5 cases and some of their family. The genetic testing revealed two pathogenic mutations (p.T309I in KCNQ1 and p.R744fs in KCNH2) and all of the mutational frequencies in KCNQ1 and KCNH2 were 20%. In the two patients who carry the pathogenic mutation presenting with recurrent syncope due to ventricular fibrillation, an implantable cardioverter defibrillator was implanted. We also discovered 11 polymorphisms in KCNQ1, 3 in KCNH2, and 5 in SCN5A. Two-fifths of cases (40%) presented with one of the three major LQTS-causing gene mutations. (Int Heart J 2015; 56: 450-453)

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“…As recently reviewed, the list of genes analyzed by HRM is increasing [13]. Among the different cardiac channelopathy susceptibility genes implicated in sudden death with structurally normal heart (namely 12 genes in LQTS [14], seven genes in Brugada Syndrome [15], two genes in CPTV [16,17]), three genes (KCNQ1, KCNH2, and SCN5A) have been studied by Millat et al by HRM analysis [12,18]. The KCNQ1, KCNH2, and SCN5A genes account for 70-75 % of definite congenital LQTS cases with a frequency in the affected people of 40-55, 35-45, and 2-8 %, respectively [14].…”

Section: Introductionmentioning

confidence: 99%

“…The KCNQ1, KCNH2, and SCN5A genes account for 70-75 % of definite congenital LQTS cases with a frequency in the affected people of 40-55, 35-45, and 2-8 %, respectively [14]. In the last study of Millat et al [18], the HRM analysis was performed on DNA extracted from whole blood in a panel of LQTS-suspected patients.…”

Section: Introductionmentioning

confidence: 99%

Detection of genetic variation in KCNQ1 gene by high-resolution melting analysis in a prospective-based series of postmortem negative sudden death: comparison of results obtained in fresh frozen and formalin-fixed paraffin-embedded tissues

Farrugia¹,

Keyser

Ludes

2012

Int J Legal Med

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High-resolution melting (HRM) analysis is a recently developed molecular technique proved to be applicable for detection of genetic variation, notably in sudden cardiac death. In certain circumstances, especially in postmortem genetic investigations, the formalin-fixed and paraffin-embedded (FFPE) tissues are the only DNA source available. The present study aimed to develop HRM assays, optimized for the analysis of FFPE tissues, to detect sequence variations in KCNQ1 exons in a prospective population-based series of postmortem negative sudden death and to compare the results between the paired freshly frozen and FFPE tissue samples simultaneously obtained from the same case. The analyses were conducted in each case of sudden death involving cases younger than 35 years with no significant morphological anomalies particularly with no cardiac structural disease and with negatives toxicological investigations. HRM analysis was successfully optimized for 13 of the 16 exons of the KCNQ1 gene. All mutated samples were correctly identified by HRM whatever the type of tissue tested. However, for FFPE samples, HRM indicated more positive samples than classical sequencing, used in parallel, due to the degradation of DNA by formalin fixation. This is the first postmortem study of KCNQ1 mutation detection with HRM on DNA extracted from FFPE samples with adapted protocol. Despite the false-positive detection, we concluded that the use of HRM as a screening method with FFPE samples to analyze KCNQ1 mutations can reduce the number of sequencing reactions and, thus, results in substantial time and cost savings.

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Development of a high resolution melting method for the detection of genetic variations in Long QT Syndrome

Cited by 11 publications

References 15 publications

L-Type Calcium Channel Mutations in Japanese Patients With Inherited Arrhythmias

L-Type Calcium Channel Mutations in Japanese Patients With Inherited Arrhythmias

Mutation Analysis of KCNQ1, KCNH2 and SCN5A Genes in Taiwanese Long QT Syndrome Patients

Detection of genetic variation in KCNQ1 gene by high-resolution melting analysis in a prospective-based series of postmortem negative sudden death: comparison of results obtained in fresh frozen and formalin-fixed paraffin-embedded tissues

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